Synthesis of udp-glucose: n-acylsphingosine glucosyltransferase inhibitors

ABSTRACT

Disclosed is a novel enantiomeric synthesis ceramide-like inhibitors of UDP-glucose: N-acylsphingosine glucosyltransferase. Also disclosed are novel intermediates formed during the synthesis.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/916,824, filed Aug. 12, 2004, which is a divisional of U.S. patentapplication Ser. No. 10/197,227, filed Jul. 16, 2002, which claims thebenefit of U.S. Provisional Application No. 60/305,814, filed Jul. 16,2001. The entire teachings of these applications are incorporated hereinby reference.

BACKGROUND OF THE INVENTION

Glycosphingolipids (GSLs) are a class of naturally occurring compoundswhich have a multitude of biological functions, including the ability topromote cell growth, cell differentiation, adhesion between cells orbetween cells and matrix proteins, binding of microorganisms and virusesto cells, and metastasis of tumor cells. GSLs are derived fromglucosylceramide (GlcCer), which is produced from ceramide andUDP-glucose by the enzyme UDP-glucose: N-acylsphingosineglucosyltransferase (GlcCer synthase). The structure of ceramide isshown below:

The accumulation of GSLs has been linked to a number of diseases,including Tay-Sachs, Gaucher's, and Fabry's diseases (see, for example,U.S. Pat. No. 6,051,598). GSLs have also been linked to certain cancers.For example, it has been found that certain GSLs occur only in tumors orat abnormally high concentrations in tumors; exert marked stimulatory orinhibitory actions on tumor growth when added to tumor cells in culturemedia; and inhibit the body's normal immunodefense system when shed bytumors into the surrounding extracellular fluid. The composition of atumor's GSLs changes as the tumors become increasingly malignant andantibodies to certain GSLs inhibit the growth of tumors.

Compounds which inhibit GlcCer synthase can lower GSL concentrations andhave been reported to be useful for treating a subject with one of theaforementioned diseases. A number of potent inhibitors of GlcCer,referred to herein as “amino ceramide-like compounds”, are disclosed inU.S. Pat. Nos. 6,051,598, 5,952,370, 5,945,442, 5,916,911 and 6,030,995.The term “ceramide-like compounds” refers to analogs of ceramide inwhich: 1) the primary alcohol is replaced with a substituted aminogroup; and 2) the alkenyl group is replaced with an aryl group,preferably phenyl or substituted phenyl. The corresponding N-deacylatedcompounds are referred to as “sphingosine-like compounds.”

Unfortunately, known methods of preparing amino ceramide-like compoundsare poorly suited for manufacturing on an industrial scale. Because ofthe two chiral centers, most known syntheses generate fourdiastereoisomers, resulting in the need to separate diastereomers bychromatography and to isolate the desired enantiomer by crystallizationafter derivitization with optically active reagents, e.g.,dibenzoyltartaric acid isomers (see, for example, Inokuchi and Radin,Journal of Lipid Research 28:565 (1987)). Neither of the processes areamenable to large scale preparations. Enantioselective synthesis ofamino ceramide-like compounds using diastereoselective reductions havebeen reported (Mitchell, et al., J. Org. Chem. 63:8837 (1998) andNishida, et al. SYNLETT 1998:389 (1998)), but require over ten steps,some of which utilized expensive reagents such as diisobutylaluminumhydride (DIABAL) and Garner Aldehyde (tert-butyl (R)-(+)-4formyl-2,2-dimethyl-3-oxazolidine carboxylate). Thus, there is acritical need for enantioselective syntheses of amino ceramide-likecompounds which are more economical and efficient, and involve fewersteps than known syntheses.

SUMMARY OF THE INVENTION

Provided herein is an efficient, highly enantioselective synthesis ofamino ceramide-like compounds. This synthesis of amino ceramide-likecompounds involves only five steps from known compounds. For example,the ceramide-like compound designated as “Compound 5” in FIG. 2 wasproduced in an enantiomeric excess of at least 99.6% and an overallyield of 9% (see Examples 1 and 2). Novel intermediates prepared duringthe course of the synthesis are also disclosed.

The present invention is directed is a method of preparing aceramide-like compound represented by Structural Formula (I):

R₁ is a substituted or unsubstituted aromatic group; preferably, R₁ is asubstituted or unsubstituted phenyl group, more preferably phenylsubstituted in the meta/para positions with —OCH₂O—, —OCH₂CH₂O— or inthe para position with halo, lower alkyl thiol, —OH, —O(phenyl),—OCH₂(phenyl), lower alkyl, amino, lower alkyl amino, lower dialkylamino, or —O(lower alkyl);

R₂ and R₃ are independently —H, a substituted or unsubstituted aliphaticgroup or, taken together with the nitrogen atom to which they arebonded, are a substituted or unsubstituted non-aromatic heterocyclicring.

R₇ is a substituted or unsubstituted aliphatic group, preferably aC1-C30 straight chain unsubstituted aliphatic group or a C1-C30 straightchained aliphatic group substituted with one or more C1-C2 alkyl groups,more preferably an unsubstituted C1-C30 straight chain alkyl or alkenylgroup and even more preferably an unsubstituted C7-C10 or C10-C16straight chain alkyl or alkenyl group.

The method of preparing a ceramide-like compound represented byStructural Formula (I) comprises a first step whereby an amine compoundHNR₂R₃ is reacted with a cyclic starting material represented byStructural Formula (II):

The reaction between the amine compound HNR₂R₃ and the cyclic startingmaterial represented by Structural Formula (II) forms an amideintermediate represented by Structural Formula (III):

In Structural Formulas (II) and (III), R₁-R₃ arc as described forStructural Formula (I); and R₅ is a substituted or unsubstitutedaromatic group, preferably a substituted or unsubstituted phenyl group.

The method of preparing a ceramide-like compound represented byStructural Formula (I) comprises a second step whereby the amino acetalgroup in the intermediate represented by Structural Formula (IV) ishydrolyzed to form the acyclic compound represented by StructuralFormula (IV).

R₁, R₂, R₃ and R₅ in Structural Formulas (IV) are as defined inStructural Formulas (I)-(III).

The method of preparing a ceramide-like compound represented byStructural Formula (I) comprises a third step whereby the acyclicprecursor compound represented by Structural Formula (IV) is reactedwith an amide reducing agent to form a compound represented byStructural Formula (V):

R₁, R₂, R₃ and R₅ in Structural Formula (V) are as defined in StructuralFormulas (I)-(IV).

The method of preparing a ceramide-like compound represented byStructural Formula (I) comprises a fourth step whereby the—NHCH(—CH₂OH)R₅ group of the amine compound represented by StructuralFormula (V) is debenzylated to form a sphingosine-like compoundrepresented by Structural Formula (VI):

Preferably, the debenzylation is achieved by hydrogenation. R₁, R₂ andR₃ are as described for Structural Formulas (I)-(V).

The method of preparing a ceramide-like compound represented byStructural Formula (I) comprises a fifth step whereby thesphingosine-like compound represented by Structural Formula (VI) isacylated to form the ceramide-like compound represented by StructuralFormula (I).

Other embodiments of the present invention include each of theindividual reactions described above, taken separately and incombination with the other reactions.

Other embodiments of the present invention are intermediates in thepreparation of the ceramide-like compound represented by StructuralFormula (I) by the methods disclosed herein. In one example, the presentinvention is directed to an intermediate represented by StructuralFormula (VII):

R₁-R₃ and R₅ are as described above for Structural Formulas (I)-(VI);and

R₄ is H₂ or O.

In another embodiment, the present invention is directed to anintermediate represented by Structural Formula (VIII):

-   -   R₄ is H, or O; and    -   R₆ is represented by Structural Formula (IX):

Phenyl ring A in Structural Formula (IX) is substituted orunsubstituted. Preferably, however, phenyl ring A is unsubstituted.Alternatively, R₄ in Structural Formula (VIII) is H₂ and R₆ is —H.

In another embodiment, the present invention is directed to anintermediate represented by Structural Formula (X):

-   -   R₅ in Structural Formula (X) is as defined for Structural        Formula (I).

The methods of the present invention can be utilized to prepareceramide-like compounds that inhibit the enzyme GlcCer synthase in fivesteps from known starting materials. The synthesis is highly efficient,resulting in an overall yield that is generally greater than 8% and inan enantiomeric excess that is typically greater than 99%. The synthesisutilizes inexpensive reagents and therefore provides an economical routeto potent inhibitors of GlcCer synthase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing the synthesis of ceramide-like compoundsrepresented by Structural Formula (I) using the methods andintermediates disclosed herein.

FIG. 2 is a schematic showing the synthesis of ceramide-like Compound(5) using the methods disclosed herein.

FIG. 3 is a schematic showing the synthesis of ceramide-like compound(13) using the methods disclosed herein.

FIG. 4 shows the structures of Compounds (5)-(8).

DETAILED DESCRIPTION OF THE INVENTION

Described herein is a five step synthesis of amino ceramide-likecompounds from known starting materials. The synthesis begins with thepreparation of the cyclic starting material represented by StructuralFormula (II). The cyclic starting material is reacted with a suitableamine, thereby opening the lactone ring and forming the amideintermediate represented by Structural Formula (III). The amino acetalin the amide intermediate is hydrolyzed to form the acyclic compoundrepresented by Structural Formula (IV). The amide of this acycliccompound is reduced with an amide reducing agent to form an aminecompound represented by Structural Formula (V), which is in turndebenzylated to form the sphingosine-like compound represented byStructural Formula (VI). The primary amine of the sphingosine-likecompound represented by Structural Formula (VI) can then be acylated toform an amino ceramide-like compound. This synthesis is shownschematically in FIG. 1. A detailed description of each reaction in thesynthesis is provided below.

The cyclic starting material represented by Structural Formula (II) isprepared according to methods described in Alker, et al. Tetrahedron54:6089 (1998) and Harwood and Robertson, Chem. Commun. 1998:2641(1998). Specifically, (5S)-5-phenylmorpholin-2-one is reacted with at(east two equivalents and preferably from about 2.5 to about 5.0equivalents of aryl aldehyde R₁CHO under dehydrating conditions. R₁ isas defined in Structural Formula (I). “Dehydrating conditions” refer toconditions under which water is removed from the reaction mixture.Removal of water can be achieved, for example, by carrying out thereaction in presence of a reagent (a “dehydrating reagent”) that reactswith water (e.g., molecular sieves) but is substantially inert towardsthe other reagents present in the reaction mixture, or removal of watercan also be achieved by azeotroping with a solvent such as toluene.Sufficient dehydrating reagent is used to remove the two equivalents ofwater (relative to cyclic starting material) released during thereaction. The concentration of reagents if typically between about 0.01M and about 5.0 M, more typically between about 0.1 M and about 1.0 M;suitable reaction temperatures range between about 50° C. and about 150°C., preferably between about 100° C. and about 120° C.

The cyclic starting material is converted to the amide intermediaterepresented by Structural Formula (UI) by reacting the cyclic startingmaterial with the amine NHR₂R₃ under conditions suitable for amidatingan ester with an amine. Such conditions are well known in the art andare described, for example, in March, “Advanced OrganicChemistry—Reactions, Mechanisms and Structure”, Third Edition, JohnWiley & Sons, 1985, pages 375-76, and references cited therein. Althoughan excess of either reagent can be used, cyclic starting material ismore commonly the limiting reagent. Generally up to about fifteenequivalents of amine relative to cyclic starting material are used,typically up to about eight equivalents. The reaction can be done neat,however, it is more usually carried out in a aprotic, non-nucleophilicsolvent at amine concentrations as dilute as 0.01 M. Amineconcentrations are more typically, however, between about 0.4 M andabout 4.0 M. Suitable solvents include halogenated solvents such aschloroform, dichloromethane and 1,2-dichloroethane, acetonitrile,dimethylformamide (DMF), ethereal solvents such as diethyl ether,tetrahydrofuran (THF) and 1,4-dioxane and aromatic solvents such asbenzene and toluene. Suitable reaction temperatures generally range fromabout 0° C. to about 100° C., typically between about 25° C. to about35° C.

Conditions for hydrolyzing aminoacetals are known in the art and aredescribed, for example, in March, “Advanced Organic Chemistry—Reactions,Mechanisms and Structure”, Third Edition, John Wiley & Sons, 1985, pages329-32, and references cited therein. For example, the aminoacetal groupin the amide intermediate represented by Structural Formula (III) can behydrolyzed with dilute aqueous mineral acid. Suitable acids includehydrochloric acid, sulfuric acid or phosphoric acid, althoughhydrochloric is the most common choice. Organic acids such as aceticacid and sulfonic acids (e.g., methansulfonic acid, toluenesulfonicacid, trifluormethylsulfonic acid and the like) can also be used. Atleast one equivalent of acid relative to the intermediate is typicallyused, but an excess of acid is preferred to ensure complete hydrolysis,for example, excesses of at least ten fold, preferably an excess ofabout two to about three fold and more preferably between about 10-50%.The concentration of acid in the reaction mixture is generally betweenabout 0.05 M to about 1.0 M, typically between about 0.1 M and about 0.5M. An organic co-solvent miscible with water is often used to solubilizethe intermediate. Examples include alcohols such as methanol or ethanoland DMF. Common solvent ratios of organic solvent to water range betweenabout 1:1 to about 8:1. Suitable reaction temperatures range fromambient temperature to about 100° C., preferably between about 60° C. toabout 80° C. Alternatively, the amino acetal can be hydrolyzed withLewis acids such as trimethylsilyl iodide, wet silica gel or LiBF₄ inwet acetonitrile, as described in March, supra.

An “amide reducing agent” is a reagent which can reduce an amide to anamine. Such reagents are known in the art and are disclosed in, forexample, in March, “Advanced Organic Chemistry—Reactions, Mechanisms andStructure”, Third Edition, John Wiley & Sons, 1985, pages 1099-1100,Brown and Krishnamurthy, Aldrichimica Acta 12:3 (1979) and referencescited therein. Examples include lithium aluminum hydride, lithiumtriethyl borohydride, borane reagents (e.g., borane.tetrahydrofuran,borane.methyl sulfide, disiamylborane, and the like), aluminum hydride,lithium trimethoxy aluminum hydride and triethyloxoniumfluoroborate/sodium borohydride. In the method of the present invention,lithium aluminum hydride is the most commonly used amide reducing agent.Although as little as 0.5 equivalents of lithium aluminum hydriderelative to amide starting material can be used, it is more common touse an excess, often up to about five equivalents. Preferably, betweenabout 1.5 and about 2.5 equivalents of lithium aluminum hydride are usedrelative to amine starting material. Ethereal solvents are typicallyused for the reduction; examples include diethyl ether, THF, glyme,diglyme and 1,4-dioxane. Suitable concentrations of reducing agent aregenerally between about 0.1 M and about 5.0 M, more typically betweenabout 0.8 M and about 1.5 M. The reduction is most commonly carried outat ambient temperature, but temperatures between about 0° C. and about80° C. or 100° C. can also be used.

To form the sphingosine-like compound represented by Structural Formula(VI), the amine compound represented by Structural Formula (V) isdebenzylated. The term “debenzylating” is used herein to refer tocleaving the carbon-nitrogen bond of a group —NH—CH₂Z, wherein Z is anaryl group, preferably phenyl. Optionally, the methylene group can bereplaced with a methine group. With respect to the sphingosine-likecompound represented by Structural Formula (VI), “debenzylation” refersto converting the —NHCH(—CH₂OH)R₅ group to —NH₂, Debenzylationconditions are well known in the art and are disclosed, for example, inGreene and Wuts, “Protective Groups in Organic Synthesis”, John Wiley &Sons (1991), pages 384-86 and references cited therein.

Preferably, debenzylation is achieved by hydrogenation under a hydrogenatmosphere and in the presence of a hydrogenation catalyst. Suitablehydrogen pressures are generally between about atmospheric pressure andabout 1000 pounds per square inch. Other sources of hydrogen (e.g.,formic acid, ammonium formate, cyclohexene and the like) can also beused. Suitable hydrogenation catalysts include 20% palladium hydroxideon carbon (Perlman's catalyst), palladium chloride, palladium, platinumoxide and palladium on carbon. Typically, between about 10% and about100% weight/weigh (w/w) relative to amine compound is used. In mostinstances, an organic acid such as formic acid, acetic acid ortrifluoroacetic acid or an inorganic acid such as hydrochloric acid orsulfuric acid is present, for example, between about one to about fiveequivalents relative to amine compound, preferably between about 1.6 toabout 2.4 equivalents. The reaction is most commonly carried out in analcoholic solvent such as methanol or ethanol with water as co-solvent(e.g., between 0% and about 50% volume/volume (v/v), preferably betweenabout 5% and about 15% v/v). Reaction temperatures between about 0° C.and about 50° C. are suitable, preferably between about 25° C. and about40° C.

Many debenzylation conditions other than hydrogenation are known in theart and are included in the present invention. Examples include sodiummetal and NH₃ (see, for example, du Vigneaud and Behrens, J. Biol. Chem.117:27 (1937)), CCl₃CH₂OCOCl, CH₃CN (see, for example, Rawal, et al., J.Org. Chem., 52:19 (1987)), Me₃SiCH₂CH₂OCOCl, THF, −50° C., then 25° C.overnight (see, for example, Campbell, et al. Tetrahedron Lett., 28:2331(1987)), α-chloroethyl chloroformate and sodium hydroxide (see, forexample Olofson, et al., J. Org. Chem. 49:2081 (1984) and DeShong andKell, Tetrahedron Lett., 27:3979 (1986)), vinyl chloroformate (see, forexample, Olofson et al., Tetrahedron Lett., 1977:1567 (1977) and Cooleyand Evain, Synthesis, 1989:1 (1989)), RuO₄, NH₃, H₂O (see, for example,Gao and Jones, J. Am. Chem. Soc., 109:1275 (1987)) andm-chloroperoxybenzoic acid followed by FeCl₂, −10° C. (see, for example,Monkovic, et al., Synthesis, 1985:770 (1985).

The sphingosine-like compound represented by Structural Formula (VI) isconverted to a ceramide-like compound by acylating the free amine.Acylations of amine groups are well known in the art and can be carriedout, for example, by reacting the amine with an acylating agentR₇C(O)—X. R₇ is as described above for Structural Formula (I) and X is aleaving group that is readily displaced by a primary amine. Conditionsfor this reaction are described in, for example, in March, “AdvancedOrganic Chemistry—Reactions, Mechanisms and Structure”, Third Edition,John Wiley & Sons, 1985 and references cited therein. Examples ofsuitable acylating agents include acid halides, anhydrides or esters.Preferably, the amine is acylated with an acid chloride. Generally,equimolar amounts of the sphingosine-like compound and the acid chlorideare used in the presence of a small excess, relative to the acidchloride, of a tertiary amine such as triethylamine,diisopropylethylamine, dimethylaminopyridine or pyridine is used.However, an excess of acid chloride (typically about 10-50%) can be usedwhen the sphingosine-like compound is limiting, and vice versa. Theconcentrations of the reagents in the reaction mixture normally varybetween about 0.005 M and about 5.0 M, and are preferably between about0.05 M and about 0.5 M. The excess of amine base can be greater thanabout 100%, but is typically between about 5% and about 25%. Aproticsolvents such as halogenated solvents are preferred (e.g., chloroform,methylene chloride and 1,2-dichloromethane), however other aproticsolvents such as ethereal solvents and hydrocarbon solvents can besuitable substitutes. Ambient temperature is normally preferred for thereaction, but temperatures between about 0° C. and about 50° can also beused.

Alternatively, the acylating agent is an activated ester R₇C(O)—OX′,wherein —OX′ is readily displaced by a primary amine. Methods ofacylating an amine with activated esters are known in the art and aredescribed in, for example, March, “Advanced Organic Chemistry—Reactions,Mechanisms and Structure”, Third Edition, John Wiley & Sons, 1985, pages371-375, and references cited therein. Many activated esters are stableenough to be isolated. N-Hydroxy succinimidyl esters, some of which arecommercially available from Aldrich Chemical Co., Milwaukee, Wis., areone example of activated esters of this type. Conditions suitable forforming an amide with an acid chloride acylating agent, described in theprior paragraph, can typically be used with a stable activated ester. Incontrast with acid chlorides, which require activation with tertiaryamines, activated esters are reactive enough so that they form amidesdirectly in the presence of primary amines. Therefore, the tertiaryamine can be omitted from the acylation reaction when activated estersare used.

Alternatively, an activated ester is formed in situ. Formation of anactivated ester in situ requires a “coupling agent”, which is a reagentthat replaces the hydroxyl group of a carboxyl acid with a group whichis susceptible to nucleophilic displacement.

Examples of coupling agents include 1,1′-carbonyldiimidazole (CDI),isobutyl chloroformate, dimethylaminopropylethyl-carbodiimide (EDC),dicyclohexyl carbodiimide (DCC). When amidating by in situ generation ofan activated ester, an excess of either the carboxylic acid or amine canbe used (typically a 50% excess, more typically about a 10-15% excess).However, it is more common when carrying out the present invention touse the amine compound as the limiting reagent. Generally, from about1.0 mole to about 10 moles of coupling agent are used per mole ofcarboxylic acid, preferably from about 1.0 mole to about 1.5 moles ofcoupling agent per mole of carboxylic acid. The reaction is generallycarried out in aprotic solvents, for example, halogenated solvents suchas methylene chloride, dichloroethane and chloroform, ethereal solventstetrahydrofuran, 1,4-dioxane and diethyl ether and dimethylformamide.Suitable reaction temperatures generally range from between about 0° toabout 100° C., but the reaction is preferably carried out at ambienttemperature.

Examples of specific conditions for carrying out the reactions describedherein are provided in Examples 1 and 2.

By utilizing the enantiomer of the compound represented by StructuralFormula (II) as the cyclic starting material, the enantiomer of thecompounds represented by Structural Formulas (III)-(VI) and (I) can beprepared by utilizing the methods described herein. The enantiomer ofthe cyclic starting material represented by Structural Formula (III) canbe prepared by reacting (5R)-5-phenylmorpholin-2-one with twoequivalents of the aldehyde R₁CHO under dehydrating conditions, asdescribed above. The enantiomer of compounds represented by StructuralFormula (III), (VII), (VIII) and (X) and methods of preparing theenantiomers of the compounds represented by Structural Formulas(II)-(VI) and (I) using procedures disclosed herein are encompassedwithin the present invention.

The term “enantiomer” as it used herein, and structural formulasdepicting an enantiomer are meant to include the “pure” enantiomer freefrom its optical isomer as well as mixtures of the enantiomer and itsoptical isomer in which the enantiomer is present in an enantiomericexcess, e.g., at least 10%, 25%, 50%, 75%, 90%, 95%, 98%, or 99%enantiomeric excess.

With regard to the variables R₁-R₅ in Structural Formulas (I)-(IX), an“aliphatic group” is non-aromatic, consists solely of carbon andhydrogen and may optionally contain one or more units of unsaturation,e.g., double and/or triple bonds. An aliphatic group may be straightchained, branched or cyclic. When straight chained or branched, analiphatic group typically contains between about 1 and about 30 carbonatoms, more typically between about 1 and about 24 carbon atoms. Whencyclic, an aliphatic group typically contains between about 3 and about10 carbon atoms, more typically between about 3 and about 7 carbonatoms. Aliphatic groups are preferably lower alkyl groups, which includeC1-30 straight chained or branched saturated hydrocarbons, preferablyC1-C24 straight chained or branched saturated hydrocarbons. Examplesinclude methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl andtert-butyl.

Aromatic groups include carbocyclic aromatic groups such as phenyl,1-naphthyl, 2-naphthyl, 1-anthracyl and 2-anthacyl, and heterocyclicaromatic groups such as N-imidazolyl, 2-imidazole, 2-thienyl, 3-thienyl,2-furanyl, 3-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 2-pyranyl, 3-pyranyl, 3-pyrazolyl, 4-pyrazolyl,5-pyrazolyl, 2-pyrazinyl, 2-thiazole, 4-thiazole, 5-thiazole,2-oxazolyl, 4-oxazolyl and 5-oxazolyl.

Aromatic groups also include fused polycyclic aromatic ring systems inwhich a carbocyclic aromatic ring or heteroaryl ring is fused to one ormore other heteroaryl rings. Examples include 2-benzothienyl,3-benzothienyl, 2-benzofuranyl, 3-benzofuranyl, 2-indolyl, 3-indolyl,2-quinolinyl, 3-quinolinyl, 2-benzothiazole, 2-benzooxazole,2-benzimidazole, 2-quinolinyl, 3-quinolinyl, 1-isoquinolinyl,3-quinolinyl, 1-isoindolyl and 3-isoindolyl.

Non-aromatic heterocyclic rings are non-aromatic carbocyclic rings whichinclude one or more heteroatoms such as nitrogen, oxygen or sulfur inthe ring. The ring can be five, six, seven or eight-membered. Examplesinclude morpholinyl, thiomorpholinyl, pyrrolidinyl, piperazinyl,piperidinyl, azetidinyl, azacycloheptyl, or N-phenylpiperazinyl.

Suitable substituents on a lower alkyl, aliphatic, aromatic,non-aromatic, heterocyclic or benzyl group are those which do notsubstantially interfere with the reactions described herein.“Interfering with a reaction” refers to substantially decreasing theyield (e.g., a decrease of greater than 50%) or causing a substantialamount of by-product formation (e.g., where by-products represent atleast 50% of the theoretical yield). Interfering substituents can beused, provided that they are first converted to a protected form.Suitable protecting groups are known in the art and are disclosed, forexample, in Greene and Wuts, “Protective Groups in Organic Synthesis”,John Wiley & Sons (1991).

Suitable substituents on an alkyl, aliphatic, aromatic, non-aromaticheterocyclic ring or benzyl group include, for example, halogen (—Br,—Cl, —I and —F), —OR, —CN, —NO₂, —NR₂, —COOR, —CONR₂, —SO_(k)R (k is 0,1 or 2) and —NH—C(═NH)—NH₂. Each R is independently —H, an aliphaticgroup, a substituted aliphatic group, a benzyl group, a substitutedbenzyl group, an aromatic group or a substituted aromatic group, andpreferably —H, a lower alkyl group, a benzylic group or a phenyl group.A substituted non-aromatic heterocyclic ring, benzylic group or aromaticgroup can also have an aliphatic or substituted aliphatic group as asubstituent. A substituted alkyl or aliphatic group can also have anon-aromatic heterocyclic ring, benzyl, substituted benzyl, aromatic orsubstituted aromatic group as a substituent. A substituted alkyl,substituted aliphatic, substituted non-aromatic heterocyclic,substituted aromatic or substituted benzyl group can have more than onesubstituent.

When R₁ is a substituted phenyl group, examples of preferredsubstitutents include —OCH₂O—, —OCH₂CH₂O—, halo, (lower alkyl)O—, loweralkyl thiol, lower dialkylamine, —OH, —O(phenyl), —OCH₂ (phenyl), loweralkyl, amine and lower alkyl amino.

When R₅ is a substituted phenyl group, examples of preferredsubstitutents include halo, (lower alkyl)O—, —O(phenyl) and lower alkyl.

In the structural formulas depicted herein, the remainder of themolecule or compound to which a chemical group or moiety is connected isindicated by the following symbol:

For example, the corresponding symbol in Structural Formula (IX)indicates that the depicted group, which is represented by R₆ inStructural Formula (VIII), is connected via the benzylic carbon to theamine in Structural Formula (VIII) by a single covalent bond.

In preferred embodiments of the present invention the variables usedherein are defined as follows: R₁ is a substituted or unsubstitutedphenyl group; R₂ and R₃ are independently —H, an unsubstituted C1-C5alkyl group or, taken together with the nitrogen atom to which they arebonded, are an unsubstituted C3-C10 non-aromatic heterocyclic ring; R₅is a substituted or unsubstituted phenyl group, preferably phenyl; andR₇ is a C1-C30 straight chain unsubstituted aliphatic group or a C1-C30straight chained aliphatic group substituted with one or more C1-C2alkyl group and more preferably an unsubstituted C1-C30 straight chainalkyl or alkenyl group.

In another preferred embodiment, —NR₂R₃, taken together, ispyrrolidinyl. More preferably, —NR₂R₃, taken together, is pyrrolidinyland R₅ is phenyl in compounds comprising R₂, R₃ and R₅. Even morepreferably, in compounds comprising R₁, R₂, R₃ and R₅, R₁ is asubstituted or unsubstituted phenyl group (preferably phenyl substitutedin the meta/para positions with —OCH₂O—, —OCH₂CH₂O— or in the paraposition with halo, lower alkyl thiol, —OH, —O(phenyl), —O—CH₂(phenyl),lower alkyl, amino, lower alkyl amine, lower dialkyl amino, or —O(loweralkyl), —NR₂R₃, taken together, is pyrrolidinyl and R₅ is phenyl.

In another preferred embodiment, —NR₂R₃, taken together, is piperidyl.More preferably, —NR₂R₃, taken together, is piperidyl and R₅ is phenylin compounds comprising R₂, R₃ and R₅. Even more preferably, incompounds comprising R₁, R₂, R₃ and R₅, R₁ is a substituted orunsubstituted phenyl group (preferably phenyl substituted in themeta/para positions with —OCH₂O—, —OCH₂CH₂O— or in the para positionwith halo, lower alkyl thiol, —OH, —O(phenyl), —OCH₂-(phenyl), —OCH₂(phenyl), lower alkyl, amino, lower alkyl amino, lower dialkyl amino, or—O(lower alkyl), —NR₂R₃, taken together, is piperidyl and R₅ is phenyl.

Examples of ceramide-like compounds which can be prepared by the methodsof the present invention are represented by Structural Formula (XI):

R₁ is phenyl substituted in the meta/para positions with —OCH₂O— or—OCH₂CH₂O— or in the para position with halo, CH₃O—, CH₃CH₂O—,CH₃CH₂CH₂O—, CH₃(CH₃)CHO—, CH₃—, CH₃CH₂—, CH₃CH₂CH₂—, CH₃(CH₃)CH—, —OHor —OCH₂(phenyl), and R₇ is CH₃(CH₂)_(n)— or CH₃(CH₂)_(n-2)CH═CH—,wherein n is an integer from 0 to about 30. Preferably, n is 7, 8,9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24. Morepreferably, R₁ is phenyl substituted meta/para with —OCH₂CH₂O—.

Compounds of this invention which possess a sufficiently acidic, asufficiently basic, or both functional groups, and accordingly can reactwith any of a number of inorganic bases, and inorganic and organicacids, to form a salt. Thus, the present invention also includes saltsof the intermediates represented by Structural Formulas (VII), (VIII)and (X). Physiologically acceptable salts are preferred. Acids commonlyemployed to form acid addition salts are inorganic acids such ashydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid,phosphoric acid, and the like, and organic acids such asp-toluenesulfonic acid, methanesulfonic acid, oxalic acid,p-bromophenyl-sulfonic acid, carbonic acid, succinic acid, citric acid,benzoic acid, acetic acid, and the like. Examples of such salts includethe sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, acetate, propionate,decanoate, caprylate, acrylate, formate, isobutyrate, caproate,heptanoate, propiolate, oxalate, malonate, succinate, suberate,sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate,phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate,gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate,propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,mandelate, and the like.

Base addition salts include those derived from inorganic bases, such asammonium or alkali or alkaline earth metal hydroxides, carbonates,bicarbonates, and the like. Such bases useful in preparing the salts ofthis invention thus include sodium hydroxide, potassium hydroxide,ammonium hydroxide, potassium carbonate, and the like.

The entire teachings of the publications cited in this application areincorporated herein by reference.

EXEMPLIFICATION Example 1—Small Scale Preparation of Ceramid-LikeCompounds Intermediate 1(1R,3S,5S,8aS)-1,3-Bis-(2′,3′-dihydro-benzo[1,4]dioxin-6′-yl)-5-phenyl-tetrahydro-oxazolo[4,3-c][1,4]oxaza-8-one

To a stirred solution of (5S)-5-phenylmorpholim-2-one (2.00 g, 11.3mmol) (prepared as in: Dellaria, J. F.: Santarsiero, B. D. J. Org.Chem., 1989, 54, 3916) and 1,4-benzodioxan-6-carboxaldehyde (5.56 g,33.9 mmol) in toluene (125 mL) was added 4 Å molecular sieves(approximately 20 mL). The mixture was heated at reflux for 72 hours,filtered free of sieves and concentrated. The resulting amber gum wasflash chromatographed over silica (diethyl ether/hexane) to furnish apale yellow solid. This material was further purified by triturationwith diethyl ether to afford 1.89 g (34%) product as a fluffy whitesolid: ¹H NMR (CDCl₃) δ 7.31-7.17 (m, 5H), 6.95-6.79 (m, 5H), 5.32-5.27(m, 2H), 4.43-4.28 (m, 2H), 4.24 (s, 4H), 4.18 (m, 4H), 4.16-4.08 (m,2H) ppm.

Intermediate 2(2S,3R,1″S)-3-(2′,3′-Dihydro-benzo[1,4]dioxin-6′-yl)-3-hydroxy-2-(2″-hydroxy-1″-phenyl-ethylamino)-1-pyrrolidin-1-yl-propan-1-one

To a stirred solution of Intermediate 1 (1.80 g, 3.69 mmol) inchloroform (20 mL) was added pyrrolidine (2.0 mL, 24 mmol). The solutionwas stirred overnight and then concentrated. The resulting colorlesstacky foam was taken up in methanol (16 mL) and 1 N hydrochloric acid (4mL). The mixture was refluxed for 1 hour, treated with additional 1 Nhydrochloric acid (2 mL) and refluxed for another 2 hours. The reactionsolution was concentrated and the resulting residue partitioned betweenethyl acetate and aqueous sodium bicarbonate solution. The organic layerwas dried (sodium sulfate) and concentrated. The resulting pale yellowgum was purified by flash chromatography over silica gel (methylenechloride/2 N methanolic ammonia) to afford 1.40 g (92%) of Intermediate2 as a colorless foamy solid: ¹H NMR (CDCl₃) δ 7.31-7.13 (m, 5H),6.93-6.70 (m, 3H), 4.47 (d, J=8.5, 1 H), 4.18 (s, 4H), 3.82 (t, J 5.9,1H), 3.74 (d, J=6.0, 2H), 3.06 (d, J=8.5, 1H), 3.06-2.97 (m, 1H),2.92-2.83 (m, 1H), 1.97-1.87 (m, 1H), 1.45-1.15 (m, 4H) ppm.

Intermediate 3(1R,2R,1″S)-1-(2′,3′-Dihydro-benzo[1,4]dioxin-6′-yl)-2-(2″-hydroxy-1″-phenyl-ethylamino)-3-pyrrolidi-1-yl-propan-1-ol

To a stirred solution of Intermediate 2 (1.38 g, 3.35 mmol) intetrahydrofuran (30 mL) was added lithium aluminum hydride (0.26 g, 6.9mmol). The foamy suspension was stirred overnight and then quenched withthe addition (dropwise until frothing ceases) of 1 N aqueous sodiumhydroxide (13 mL). The mixture was diluted with water and extracted withethyl acetate. The organic layer was dried (sodium sulfate) andconcentrated to afford a colorless gum. Flash chromatography over silicagel (methylene chloride/2 N methanolic ammonia) afforded 0.94 g (70%) ofproduct as a colorless tacky foam: ¹H NMR (CDCl₃) δ 7.36-7.17 (m, 5H),6.88-6.74 (m, 3H), 4.42 (d, J=5.4, 1H), 4.26 (s, 4H), 3.79-3.69 (m, 1H),3.64-3.56 (m, 1H), 3.55-3.45 (m, 1H), 3.00-2.90 (m, 1H), 2.67-2.57 (m,1H), 2.43-2.32 (m, 4H), 2.25-2.15 (m, 1H), 1.75-1.65 (m, 4H) ppm.

Intermediate 4(1R,2R)-2-Amino-1-(2′,3′-dihydro-benzo[1,4]dioxin-6′-yl)-3-pyrrolidi-1yl-propan-1-ol

In a high pressure reaction bomb equipped with a mechanical stirrer wasloaded a solution of Intermediate 3 (0.91 g, 2.28 mmol) in 10:1methanol/water (22 mL), trifluoroacetic acid (0.18 mL, 2.3 mmol) and 20%palladium hydroxide on carbon (Perlman's catalyst; 0.91 g). The reactorwas evacuated and backfilled with argon three times and then evacuatedand refilled with hydrogen (100 psi). The reaction was stirred for 2days and then evacuated and flushed with nitrogen. The reaction solutionwas filtered through Celite and concentrated. The resulting gray-greengum was flash chromatographed over silica gel (methylene chloride/2 Nmethanolic ammonia) to afford 0.165 g (26%) of product as a nearcolorless gum: ¹H NMR (CDCl₃) δ 6.89-6.76 (m, 3H), 4.54 (d, J=3.7, 1H),4.25 (s, 4H), 3.43 (s, 1H), 3.14-3.07 (m, 1H), 2.68-2.41 (m, 6H),1.82-1.71 (m, 4H) ppm.

Compound 5 (1R,2R)-Hexadecanoic acid[2-(2′,3′-dihydro-benzo[1,4]dioxin-6′-yl)-2-hydroxy-1-pyrrolidin-1-ylmethyl-ethyl]-amide

To a stirred solution of Intermediate 4 (0.165 g, 0.593 mmol) inmethylene chloride (8 mL) was added palmitoyl chloride (0.18 g, 0.59mmol) followed by N,N-diisopropylethylamine (0.11 mL, 0.65 mmol). Thesolution was stirred for 2 hours and then concentrated. The residue waspartitioned between ethyl acetate and aqueous sodium bicarbonatesolution. The organic layer was dried (sodium sulfate) and concentrated.The resulting off-white solid was flash chromatographed over silica gel(methylene chloride/2 N methanolic ammonia) to afford 0.174 g (57%) ofproduct as a white solid. Comparisons by ¹H NMR spectroscopy andanalytical chiral HPLC (column: Chirex (S)-VAL and (R)-NE, 4.6×250 mm;eluant: 0.5% trifluoroacetic acid in 67:31:2 hexane/methylenechloride/ethanol; flow: 1 mL/min; detection 280 nM) demonstrate thatthis material was identical to a sample of the same compound prepared bythe method of Polt, et al. (J. Org. Chem., 1998, 63, 8837). Enantiomericexcess was determined to be 99.6%. Total contamination from the twopossible diastereomers is determined to be 0.2%. ¹H NMR (CDCl₃) δ6.88-6.73 (m, 3H), 5.84 (d, J=7.3, 1H), 4.90 (d, J=3.8, 1H), 4.24 (s,4H), 4.22-4.15 (m, 1H), 2.86-2.72 (m, 2H), 2.72-2.55 (m, 4H), 2.10 (t,J=7.5, 2H), 1.82-1.74 (m, 4H), 1.58-1.46 (m, 2H), 1.32-1.16 (m, 24H),0.88 (t, J=6.7, 3H) ppm.

Example 2—Large Scale Preparation of Ceramide-Like Compounds(5S)-5-Phenylmorpholin-2-One

A solution of S-(+)-Phenyl glycinol (Aldrich, 10.17 g, 78.12 mmol) andDiisopropylethylamine (Aldrich, 34 mL, 195 mmol, 2.5 equivalents) wasprepared in CH₃CN (200 mL). This solution was added tophenyl-α-bromoacetate (18.48 g, 85.9 mnol, 1.1 equivalents) dissolved inCH₃CN (50 mL) under nitrogen dropwise over 2 hours. The resultingsolution was stirred under nitrogen for 16-20 hours. The solvent wasremoved by rotoevaporation keeping the bath temperature at below 25° C.To the oil was added ethyl acetate (120 mL) and the mixture was stirredfor 15 minutes. The resulting white precipitate was filtered off and thesolid washed with ethyl acetate (25 mL). The filtrate was rotoevaporatedto an oil keeping the bath temperature below 25° C. After drying undervacuum for 0.5 hours, the oil was dissolved in CH₂Cl₂ (17 mL) and loadedonto a silica gel column (60 g packed with 10% ethyl acetate/hexanes.The upper byproduct spots were eluted with 10% ethyl acetate/hexanes andthe product was eluted with 50% ethyl acetate/hexanes −100% ethylacetate. The fractions containing the product were rotoevaporated to anoil keeping the bath temperature below 25° C. This oil was dissolved inethyl acetate (12 mL) and hexanes (60 mL) was added slowly in an icebath to precipitate the product. The resulting precipitate was filtered.The white to yellow solid was vacuum dried. The(5S)-5-phenylmorpholin-2-one obtained (7.4 g, 41.8 mmol, 53%) was useddirectly in the next step.

Intermediate 1(1R,3S,5S,8aS)-1,3-Bis-(2′,3′-dihydro-benzo[1,4]dioxin-6′-yl)-5-phenyl-tetrahydro-oxazolo[4,3-c][1,4]oxazin-8-one

(5S)-5-Phenylmorpholin-2-one (7.4 g, 41.8 mmol) andbenzodioxolane-6-carboxaldehyde (Aldrich or Alfa Aesar, 20.56 g, 125.2mmol, 3.0 equivalents) was dissolved in toluene (180 mL). The solutionwas placed in a soxhlet extractor apparatus filled with 4 Å molecularsieves (ca 30 g). The solution was refluxed under nitrogen for 2-3 days.After cooling to room temperature, the solvent was removed byrotoevaporation and the oil was dissolved in ethyl acetate (200 mL). Asolution of sodium bisulfite (Aldrich, 50 g) in water (100 mL) was addedand the two phase mixture was stirred at room temperature for 1 hour.The resulting white solid was filtered off and washed with ethylacetate. The filtrate was placed in a separatory funnel and the layersseparated. The organic layer was washed with water (100 mL) andsaturated sodium chloride solution (100 mL). The dried (Na₂SO₄) solutionwas filtered and rotoevaporated to a yellow-red foamy oil (23.11 g).After drying under vacuum for 1 hour, diethyl ether (350 ml) was addedand the mixture was stirred at room temperature for 16-20 hours. Theresulting white-yellow solid was filtered. The solid was dried undervacuum. The cycloadduct was obtained in 46% yield (9.34 g).

Intermediate 2(2S,3R,1″S)-3-(2′,3′-Dihydro-benzo[1,4]dioxin-6′-yl)-3-hydroxy-2-(2″-hydroxy-1″-phenyl-ethylamino)-1-pyrrolidin-1yl-propan-1-one

To the cycloadduct (Intermediate 1, 6.7 g, 13.74 mmol) dissolved inmethylene chloride (40 mL) was added pyrrolidine (Aldrich, 5.7 mL, 68.7mmol, 5 equivalents). The solution was stirred under nitrogen at roomtemperature for 16-18 hours. The solvent was rotoevaporated to yield ayellow foamy oil which was vacuum dried for 0.5 hours. The crude wasdissolved in methanol (115 mL) and a 1 M aqueous HCl solution (115 mL)was added. The solution was refluxed for 4 hours. After cooling to roomtemperature, the methanol was removed by rotoevaporation. Ethyl acetate(60 mL) was added and the two phase system was stirred at roomtemperature for 5-15 minutes. The two layers were separated and theorganic layer was extracted with 1 M HCL (30 mL). The combined aqueouslayers were washed two times with ethyl acetate (60, 30 mL). A saturatedsodium bicarbonate solution (150 mL) was added to the aqueous layerslowly. The product was extracted three times with ethyl acetate (60 mL)from the basic (pH=8-9) aqueous layer. The combined organic layerscontaining the product were washed with a saturated sodium chloridesolution (30 mL). After drying with Na₂SO₄ the solution was filtered androtoevaporated to yield a yellow solid. Intermediate 2 was obtained in93% yield (5.26 g).

Intermediate 3(1R,2R,1″S)-1-(2′,3′-Dihydro-benzo[1,4]dioxin-6′-yl)-2-(2″-hydroxy-1″-phenyl-ethylamino)-3-pyrrolidi-1-yl-propan-1-ol

To a 3-neck flask equipped with a dropping funnel and condenser wasadded LiAlH₄ (Aldrich, 1.2 g, 31.7 mmol, 2.5 equivalents) and anhydrousTHF (20 mL) under nitrogen. A solution of Intermediate 2 (5.23 g, 12.68mmol) in anhydrous THF (75 mL) was added dropwise to the reaction over15-30 minutes. The reaction was refluxed under nitrogen for 9 hours. Thereaction was cooled in an ice bath and a 1M NaOH solution was carefullyadded dropwise. After stirring at room temperature for 15 minutes, water(50 mL) and ethyl acetate (75 mL) was added. The layers were separatedand the aqueous layer was extracted twice with ethyl acetate (75 mL).The combined organic layers were washed with saturated sodium chloridesolution (25 mL). After drying with Na₂SO₄ the solution was filtered androtoevaporated to yield a colorless to yellow foamy oil. Intermediate 3was obtained in 99% yield (5.3 g).

Intermediate 4(1R,2R)-2-Amino-1-(2′,3′-dihydro-benzo[1,4]dioxin-6′-yl)-3-pyrrolidin-1-yl-propan-1-ol

Intermediate 3 (5.3 g, 13.3 mmol) was dissolved in methanol (60 mL).Water (6 mL) and trifluoroacetic acid (2.05 mL, 26.6 mmol, 2equivalents) were added. After being placed under nitrogen, 20%Palladium hydroxide on carbon (Pearlman's catalysis, Lancaster orAldrich, 5.3 g) was added. The mixture was placed in a Parr PressureReactor Apparatus with glass insert. The apparatus was placed undernitrogen and then under hydrogen pressure 110-120 psi. The mixture wasstirred for 2-3 days at room temperature under hydrogen pressure 100-120psi. The reaction was placed under nitrogen and filtered through a padof celite. The celite pad was washed with methanol (100 mL) and water(100 mL). The methanol was removed by rotoevaporation. The aqueous layerwas washed with ethyl acetate three times (100, 50, 50 mL). A 10 M NaOHsolution (10 mL) was added to the aqueous layer (pH=12-14). The productwas extracted from the aqueous layer three times with methylene chloride(100, 100, 50 mL). The combined organic layers were dried with Na₂SO₄,filtered and rotoevaporated to a colorless oil. The foamy oil was vacuumdried for 2h. Intermediate 4 was obtained in 90% yield (3.34 g).

Compound 5 (1R,2R)-Hexadecanoic acid[2-(2′,3′-dihydro-benzo[1,4]dioxin-6′-yl)-2-hydroxy-1-pyrrolidin-1-ylmethyl-ethyl]amide

To a solution of Intermediate 4 (3.34 g, 12.0 mmol) in methylenechloride (50 mL) was added a solution of palmitic acidN-hydroxylsuccinimide ester (Sigma, 4.24 g, 12.0 mmol) over 15-30minutes under nitrogen at room temperature. The solution was stirred atroom temperature for 18-20 hours. To the reaction was added methylenechloride (50 mL) and a 1 M NaOH solution (25 mL). The two phase systemwas stirred at room temperature for 15-30 min. Water (25 mL) was addedand the layers were separated. The aqueous layer was back extracted withmethylene chloride (25 mL). The combined organic layers were washedtwice with water (25 mL) and once with a saturated sodium chloridesolution (25 mL). The organic layer was dried with Na₂SO₄, filtered androtoevaporated to a light yellow oil. The crude was recrystallized fromhexane (50 mL). The white solid (5.46 g) obtained was separated onsilica gel (300 g) with 2% methanol; methylene chloride—4% methanol:methylene chloride—4% 2 M ammonium in methanol: methylene chloride. Thewhite solid obtained was recrystallized form hexanes (70 mL). Compound 5was obtained in 66% yield (4.18 g). Analytical chiral HPLC (column:Chirex (S)-VAL and (R)-NE, 4.6×250 mm; eluant: 0.5% trifluoroacetic acidin 67:31:2 hexane/methylene chloride/ethanol; flow: 1 mL/min; detection:280 nM) showed this material to be 98.98% pure with 0.89% of adiastereoisomer and 0.14% of the enantiomer.

Example 3—Alternative Large Scale Preparation of Ceramide-Like Compounds(5S)-5-Phenylmorpholin-2-one HCl Salt

A solution of phenyl bromoacetate (Aldrich, 862.17 g, 4.0 moles, 1.1equivalents) in acetonitrile (reagent grade, 1500 ml) was cooled in anice bath (internal temperature below 5° C.). To this was added a coldslurry (internal temperature below 5° C.) of S-(+)-2-phenyl glycinol(Aldrich, 500 g, 3.65 moles, 1 equivalent) and diisopropylethylamine(DIPEA) (Aldrich, 1587 ml, 9.11 moles, 2.5 equivalents) in acetonitrile(2900 ml) in portions while keeping the internal temperature below 10°C. The mixture was stirred at this temperature for 30 minutes before theice bath was removed and the mixture was allowed to stir at roomtemperature for an additional 4 hours. The solvent was removed in vacuowhile maintaining the bath temperature at 25° C. The mixture wascoevaporated with ethyl acetate (2×500 ml) to produce a light yellowviscous oil. To the reaction mixture, ethyl acetate (4500 ml) was addedand the flask was immersed in an ice bath with agitation. The mixturewas allowed to cool below 8° C. The solid was filtered and washed withethyl acetate (3×250 ml). The solution was cooled to below 5° C. Dry HClgas was passed slowly into the solution while maintaining the internaltemperature below 15° C. until the pH was below 2 (wet pH paper). Themixture was allowed to stir at this temperature and pH for an additional20 minutes before the solid was suction filtered. The solid was washedwith ethyl acetate (3×200 ml) and dried under high vacuum for about 20hours. The yield was 412 g (53%). ¹H NMR was consistent with the(5S)-5-phenylmorpholin-2-one HCl salt.

Intermediate 1(1R,3S,5S,8aS)-1,3-Bis-(2′,3′-dihydro-benzo[1,4]dioxin-6-yl)-S-phenyl-tetrahydro-oxazolo[4,3-c][1,4]oxazin-8-one

To a stirred suspension of (5S)-5-phenylmorpholin-2-one HCl salt (381 g,1 equivalent) in 15% ethyl acetate in toluene (2270 ml) was added asolution of sodium bicarbonate (1.1 equivalents) in water (2000 mil).The resulting biphasic solution was stirred at room temperature forabout 1 hour. The organic layer was transferred to a flask containing1,4-benzodioxan-6-carboxaldehyde. The flask was then equipped with aDean-Stark unit, a condenser and a nitrogen inlet. The mixture washeated at reflux with agitation while about 650 ml solvent (mixture ofethyl acetate and toluene) was collected via Dean-Stark unit. Theresulting yellow-red solution was allowed reflux for about 64 hours,under nitrogen while the water formed during the reaction was collectedin the Dean-Stark unit. Most of the solvent was then removed viadistillation at atmospheric pressure through Dean-Stark unit. Theresidual solvent was then removed by coevaporation with heptane (500 ml)and tert-butylmethyl ether (2×725 ml) to produce a yellow semi solidproduct. The semi solid product was dissolved in ethyl acetate (3400ml). A solution of sodium bisulfite (920 g) in water (1500 ml) was addedand the mixture was allowed to stir at room temperature for about 1hour. The solid that was formed was removed by filtration and washedwith ethyl acetate (3×400 ml). The filtrate was washed with water (1450ml), 5% brine solution (1450 ml) and dried over MgSO₄ (100 g). Thesolvent was removed in vacuo to afford a yellow solid. To this was addedtert-butylmethyl ether (2900 ml) and the suspension was stirred at roomtemperature for 20 to 22 hours. The yellow solid was suction filtered,washed with tert-butylmethyl ether (2×600 ml) and dried under highvacuum at room temperature for about 22 hours. The yield was 400.5 g(58%). ¹H NMR and TLC were consistent with Intermediate 1.

Intermediate 22S,3R,1″S)-3-(2′,3′-Dihydro-benzo[1,4]dioxin-6′-yl)-3-hydroxy-2-(2″-hydroxy-1″-phenyl-ethylamino)-1-pyrrolidin-1-yl-propan-1-one

A solution of Intermediate 1 (312 g, 0.64 moles), pyrrolidine (267 ml,3.2 moles, 5 equivalents) and tetrahydrofuran (1350 ml) was heated atreflux for 4.5 hours under nitrogen atmosphere. The solvent and excesspyrrolidine were removed in vacuo to produce the crude intermediate asan orange viscous oil. The oil was dissolved in methanol (3000 ml) and1M hydrochloric acid solution (3000 mil). The resulting solution washeated at reflux for about 7 hours. The solvent was then removed invacuo to afford a mixture of an oil and water. To this ethyl acetate(2000 ml) was added and the aqueous layer was separated. The organiclayer was extracted with aqueous 1M HCl (1000 ml). The aqueous layerswere combined and washed with ethyl acetate (2000 ml). The aqueous layerwas cooled in an ice bath. The pH of the aqueous layer was adjusted toabout 9 (pH paper) with 10 M aqueous NaOH (525 ml). The aqueous layerwas extracted with ethyl acetate (3000 ml). The organic layer was washedwith 5% brine solution (1000 ml) and dried (Na₂SO₄). The solvent wasremoved in vacuo to produce a yellow viscous oil. The yield was 213.4 g,81%. ¹H NMR was consistent with Intermediate 2.

Intermediate 31R,2R,1″S)-1-(2′,3′-Dihydro-benzo[1,4]dioxin-6′-yl)-2-(2″-hydroxy-1″-phenyl-ethylamino)-3-pyrrolidin-1-yl-propan-1-ol

To a slurry of LiAlH₄ (50.7 g, 1.34 moles, 2.6 equivalents) intetrahydrofuran (700 ml) was added a solution of Intermediate 2 (213.34g, 0.517 moles) in tetrahydrofuran (2000 ml) slowly with agitation atroom temperature. The mixture was refluxed for about 4 hours. TLCanalysis (10% methanol in methylene chloride, v/v) indicated consumptionof the starting material. The reaction mixture was cooled in an ice bath(below 5° C.) and water (135 ml) was added very slowly while keeping theinternal temperature less than or equal to 10° C. To this was then addeda 15% aqueous NaOH solution (70 ml) followed by water (200 ml). Thereaction mixture was allowed to warm to room temperature while theagitation was continued. Methylene chloride (1000 ml) was then added tothe mixture and the salts were filtered through a pad of celite. Thesalts were washed with methylene chloride (2×500 ml). The filtrates werecombined and the solvent was removed in vacuo to produce a yellow oil.The oil was dissolved in 1M aqueous HCl (1500 ml) and washed with ethylacetate (3×500 ml). The aqueous layer was cooled in an ice bath to below5° C. and the pH of the aqueous layer was adjusted to 12 to 13 with a 10M aqueous NaOH solution (220 ml) keeping the internal temperature atless than or equal to 10° C. The mixture was allowed to warm to roomtemperature. The aqueous layer was extracted with methylene chloride(2×500 ml). The organic layers were combined and washed with brinesolution (500 ml), dried (Na₂SO₄) and the solvent was removed in vacuoto afford a yellow viscous oil. The yield was 186.4 g (88.5%). ¹H NMRwas consistent with Intermediate 3.

Intermediate 4 Dioxalate Salt(1R,2R)-2-Amino-1(2′,3′-dihydro-benzo[1,4]dioxin-6′-yl)-3-pyrrolin-1-yl-propan-1-olDioxalate Salt

A suspension of Intermediate 3 (358 g, 0.90 moles), ethanol (1500 ml),1M HCl solution (1500 ml) and 10% Pd(OH)₂ (32 g, 20 weight %) werehydrogenated at about 50 psi for about 36h at room temperature. Themixture was filtered through a Cuono filter. The Cuono filter was washedwith 10% ethanol in water (500 ml). The filtrates were combined andethanol was removed in vacuo. The aqueous layer was extracted with ethylacetate (3×600 ml). The organic layer was extracted with 1M HCl aqueous(700 ml). The aqueous layers were combined and cooled in an ice bath (0about 5° C.). The pH of the aqueous layer was adjusted to about 12 (pHpaper) with 10 M aqueous NaOH solution (490 ml) keeping the internaltemperature below 10° C. The aqueous layer was allowed to warm to roomtemperature. The aqueous layer was extracted with methylene chloride(2×1500 ml, 1×750 ml). The combined organic layers were dried over MgSO₄and the solvent was removed in vacuo to afford a yellow viscous oil. Thecrude weight was 214.3 g (86%). ¹H NMR was consistent with Intermediate4.

A solution of oxalic acid (152.4 g, 1.693 moles, 2.2 equivalents) inmethylisobutyl ketone (2300 ml) was added slowly with stirring to asolution of Intermediate 4 (214.3 g, 0.77 moles, 1 equivalent) inmethylisobutyl ketone (800 ml) at room temperature. The resultingmixture was stirred at room temperature for about 2.5 hours. The solidwas filtered, and triturated with acetone (2000 ml) at room temperaturefor about 16 hours. The solid was filtered, washed with acetone (3×100ml) and dried under high vacuum to produce an off-white solid. The yieldwas 312.5 g (89%). ¹H NMR was consistent with Intermediate 4 dioxalatesalt.

Compound 5 (1R,2R)-Hexadecanoic acid[2-(2′,3′-dihydro-benzo[1,4]dioxin-6′-yl)-2-hydroxy-1-pyrrolidin-1-ylmethyl-ethyl]-amide

To a cold solution (about 5° C.) of Intermediate 4 dioxalate salt (507g, 1.11 moles) in water (10 L) was added a 10 M aqueous NaOH solution(500 ml) with stirring while keeping the internal temperature below 10°C. The solution was allowed to warm to room temperature while the pH ofthe solution was maintained at about 14 (pH paper). The aqueous layerwas extracted with methylene chloride (3×6000 ml). The organic layerswere combined, washed with water (2000 ml), dried (MgSO₄) and thesolvent was removed in vacuo to afford a yellow viscous oil,Intermediate 4. The yield was 302 g (98%). ¹H NMR was consistent withIntermediate 4.

A solution of palmitic acid NHS-ester (Sigma, 382.5 g, 1.01 equivalents)in methylene chloride (2500 ml) was added to a solution of intermediate4 (302 g) in methylene chloride (1500 ml) at room temperature over aperiod of 1.25 hours under a nitrogen atmosphere. The mixture wasallowed to stir at room temperature for about 18 hours. A solution of 1Maqueous NaOH (2425 ml) was added and the mixture was stirred at roomtemperature for about 3 hours. The organic layer was separated and theaqueous layer was extracted with methylene chloride (800 ml). Theorganic layers were combined, washed with a 1M NaOH solution (3×1500 ml)and water (1500 ml). The organic layer was dried over MgSO₄ and thesolvent was removed in vacuo to afford a semi solid. The semi-solid wascoevaporated with heptane (3×100 ml). The crude product was transferredto a 12 L three-necked RB flask and heptane (7500 ml) was added. Themixture was heated at reflux with stirring under a nitrogen atmosphere.The solution was slowly cooled to about 55° C. (internal temperature)and poured into another flask. The solution was stirred at roomtemperature for 24 hours under a nitrogen atmosphere. The off whitesolid was filtered, washed with heptane (2×500 ml) and dried under highvacuum for 24 hours. The solid (397 g) was transferred to a 12 L RBflask and 30% ethyl acetate in heptane (8000 ml) was added. Theresulting mixture was heated at reflux for 30 minutes with stirring. Thesolution was cooled to about 55° C. (internal temperature) and pouredinto another flask. The stirring was continued at room temperature undera nitrogen atmosphere for about 24 hours. The solid was filtered, washedwith heptane (2×100 ml) and dried under high vacuum to afford an offwhite solid. The yield was 324 g (58%). ¹H NMR and TLC were consistentwith Compound 5. mp 96.1° C. HPLC analysis: chiral purity 99.7%,chemical purity 99.7%. Anal. Calcd for C₃₁H N₂O₄: C, 72.05; H, 10.14; N,5.42. Found C, 72.03; H, 10.19; N, 5.42.

Example 4—Preparation of Compounds 6-8

N-hydroxysuccinimide esters of fatty acids were prepared by the methodof Lapidot, Y. Rappoport, S. and Wolman, Y. Journal of Lipid Research 8,1967 or as described below:

Octanoic Acid N-hydroxysuccinimide Ester

N-hydroxysuccinimide (Aldrich, 20.0 g, 173 mmol) and triethyl amine (29mL, 208 mmol) were dissolved in methylene chloride in an ice bath undernitrogen. Octanoyl chloride (Aldrich, 35 mL, 205 mmol) was addeddropwise over 0.5 hours. The ice bath was removed and the solution witha white solid was stirred for 1 hour at room temperature. The whitesolid was removed by filtration and the filtrate was washed with water(100 mL) and saturated aqueous sodium bicarbonate (100 mL). The organiclayer was dried with sodium sulfate, filtered and heptane (100 mL) wasadded. The solution was rotoevaporated to remove most of the methylenechloride and leave a colorless to white flaky precipitate in heptane.The precipitate was filtered and washed with heptane. After drying,Octanoic acid N-hydroxysuccinimide ester was obtained in 84% yield (35.4g).: ¹H NMR (CDCl₃) 2.84 (br s, 4H), 2.60 (t, J=7.48 Hz, 2H), 1.78-1.71(m, 2H), 1.42-1.26 (m, 8H), 0.88 (t, J=6.7 Hz, 3H) ppm.

Compound 6 (1R,2R)-Octanoic acid[2-(2′,3′-dihydro-benzo[1,4]dioxin-6′-yl)-2-hydroxy-1-pyrrolidin-1-ylmethyl-ethyl]-amide

To Intermediate 5 (22.36 g, 80.33 mmol) dissolved in anhydrous methylenechloride (300 mL) was added a solution of octanoic acidN-hydroxysuccinimide ester (19.4 g, 80.39 mmol) dissolved in anhydrousmethylene chloride (150 mL) over 15-30 minutes under nitrogen at roomtemperature. The solution was stirred at room temperature for 18-20hours. To the reaction was added 1M aqueous NaOH solution (200 mL). Thetwo phase system was stirred at room temperature for 45 minutes. Thelayers were separated and the combined organic layers were washed twicewith 1 M NaOH (2×200 mL) and twice with water (2×100 mL). The organiclayer was dried with sodium sulfate, filtered and rotoevaporated to ayellow oil. Most of the crude material was dissolved in 5% ethyl acetatein heptane (1 L) at reflux. After cooling to 40° C., the hazy solutionwas separated from the yellow oil by decanting the solution into a newflask. The first flask was rinsed twice with 5% ethyl acetate in heptane(2×250 mL) by the same process (reflux and cooling to 40° C. anddecanting the solution from the oil). The combined solution was heatedto reflux and allowed to cool to room temperature over 4 hours. Theresulting white solid was filtered and washed with 5% ethyl acetate inheptane (100 mL) and heptane (100 mL). The white solid (13.9 g) wasdried under vacuum for 16-24 hours. This solid was mostly dissolved in5% ethyl acetate in heptane (800 mL) at reflux. After cooling to 50° C.,the hazy solution was separated from the yellow oil by decanting thesolution into a new flask. The first flask was rinsed with 5% ethylacetate in heptane (100 mL) by the same process (reflux and cooling to50° C. and decanting the solution from the oil). The combined solutionwas heated to reflux and allowed to cool to room temperature over 4hours. The resulting white solid was filtered and washed with 5% ethylacetate/heptane (50 mL) and heptane (50 mL). After drying at roomtemperature under vacuum for 2-3 days, Compound 6 was obtained in 39%yield (12.57 g). Analytical chiral HPLC (column: Chirex (S)-VAL and(R)-NE, 4.6×250 mm) showed this material to be 99.9% the desired R,Risomer. Analytical HPLC showed this material to be 99.6% pure. mp 87-88°C. ¹H NMR (CDCl₃) δ 6.86-6.73 (m, 3H), 5.84 (d, J=7.3 Hz, 1H), 4.91 (d,J=3.4 Hz, 1H), 4.25 (s, 4H), 4.24-4.18 (m, 1H), 2.85-2.75 (m, 2H),2.69-2.62 (m, 4H), 2.10 (t, J=7.3 Hz, 2H), 1.55-1.45 (m, 2H), 1.70-1.85(m, 4H), 1.30-1.15 (m, 8H), 0.87 (t, J=6.9 Hz, 3H) ppm.

Compound 7 (1R,2R)-Nonanoic acid[2-(2′3′-dihydro-benzo[1,4]dioxin-6′-yl)-2-hydroxy-1-pyrrolidin-1-ylmethyl-ethyl]-amide

This compound was prepared by the method described for Compound 6 usingNonanoic acid N-hydroxysuccinimide ester. Analytical HPLC showed thismaterial to be 98.4% pure. mp 74-75° C. ¹H NMR (CDCl₃) δ 6.86-6.76 (m,3H), 5.83 (d, J=7.3 Hz, 1H), 4.90 (d, J=3.3 Hz, 1H), 4.24 (s, 4H),4.24-4.18 (m, 1H), 2.85-2.75 (m, 2H), 2.69-2.62 (m, 4H), 2.10 (1, J=7.3Hz, 2H), 1.55-1.45 (m, 2H), 1.70-1.85 (m, 4H), 1.30-1.15 (m, 10H), 0.87(t, J=6.9 Hz, 3H) ppm.

Compound 8(1R,2R)-Decanoic[2-(2′,3′-dihydro-benzo[1,4]dioxin-6′-yl)-2-hydroxy-1-pyrrolidin-1-ylmethyl-ethyl]-amide

This compound was prepared by the method described for Compound 6 usingdecanoic acid N-hydroxysuccinimide ester. Analytical HPLC showed thismaterial to be 99.3% pure. mp 97.5-98.5° C. ¹H NMR (CDCl₃) δ 6.86-6.76(m, 3H), 5.83 (d, J=7.5 Hz, 1H), 4.90 (d, J=3.4 Hz, 1H), 4.24 (s, 4H),4.24-4.18 (m, 1H), 2.85-2.75 (m, 2H), 2.69-2.62 (m, 4H), 2.10 (t, J=7.5Hz, 2H), 1.55-1.45 (m, 2H), 1.70-1.85 (m, 4H), 1.30-1.15 (m, 12H), 0.87(t, J=6.8 Hz, 3H) ppm.

Example 5—Preparation of Compound 13 Intermediate 9(1R,3S,5S,3aS)-1,3-Bis-(4-benzyloxy-phenyl)-5-phenyl-tetrahydro-oxazolo[4,3-c][1,4]oxazin-8-one

The (5S)-5-phenylmorpholin-2-one.HCl salt (57.45, 268.9 mmol) wasstirred with ethyl acetate (500 mL) and saturated aqueous sodiumbicarbonate (250 mL) for 30 minutes, until the biphasic solution wasclear. The phases were separated, and the aqueous layer was extractedwith ethyl acetate (2×250 mL). The combined organic phases were washedwith saturated sodium chloride solution (250 mL). The organic layer wasdried with sodium sulfate, filtered, concentrated to an oil, and driedunder vacuum for 60 minutes. The 5-(S)-phenyl morpholin-2-one wasobtained in a 86% yield (40.98 g, 231.3 mmol).

The 5-(S)-phenyl morpholin-2-one (40.98 g, 231.3 mmol) and4-benzyloxybenzaldehyde (Aldrich, 147.3 g, 694 mmol, 3.0 equivalents)was dissolved in toluene (750 mL). The reaction was fitted with a DeanStark Trap and a reflux condenser. The solution was refluxed undernitrogen for 2 days. After cooling to room temperature, the solvent wasremoved by rotoevaporation and the oil was dissolved in ethyl acetate(500 mL). A solution of sodium bisulfite (Aldrich, 125 g) dissolved inwater (250 mL) was added and the two phase mixture was stirred at roomtemperature for 3 hours. The resulting white solid was filtered off andwashed with ethyl acetate. The filtrate was placed in a separatoryfunnel and the layers separated. The organic layer was washed with water(250 mL), saturated aqueous sodium chloride solution (250 mL) and thendried (sodium sulfate) filtered and rotoevaporated to a foamy oil (144g). After drying under vacuum for 1 hour, tert-butyl methyl ether (1450mL) was added and the mixture was stirred at room temperature for 5hours. The resulting white-yellow solid was filtered. The solid wasdried under vacuum. Intermediate 9 was obtained in 27% yield (41.64 g,71.46 mmol). ¹H NMR (CDCl₃) δ 7.5-6.8 (m, 23H), 5.0 and 5.1 (2 s, 4H),4.5-4.3 (m, 2H), 4.2-4.1 (m, 2H) ppm.

Intermediate 10(2S,3R,1″S)-3-(4-Benzyloxy-phenyl)-3-hydroxy-2-(2″-hydroxy-1″-phenyl-ethylamino)-1-pyrrolidin-1-yl-propan-1-one

To Intermediate 9 (45.1 g, 77.4 mmol) dissolved in tetrahydrofuran (250mL) was added pyrrolidine (Aldrich 33 mL, 395 mmol, 5.1 equivalents).The solution was stirred capped under nitrogen at room temperature for16-18 hours. The solvent was rotoevaporated to yield a yellow foamy oilwhich was vacuum dried for 0.5 hours. The crude was dissolved inmethanol (220 mL) and a 1M aqueous HCl solution (220 mL) was added. Thesolution was refluxed for 4 hours. After cooling to room temperature,the methanol was removed by rotoevaporation. To the resulting oil wasslowly added 10 M aqueous NaOH (22 mL to adjust the pH to 14). Theproduct was extracted three times with methylene chloride (300,100,100mL) from the basic aqueous layer. After drying with sodium sulfate thecombined organic layer was filtered and rotoevaporated to yield ayellow-orange foamy solid. Tert-butyl methyl ether (300 mL) was addedand the mixture was stirred at room temperature for 7 hours. Theresulting white-yellow solid was filtered, washed with tert-butyl methylether (50 mL) and vacuum dried. Intermediate 10 was obtained in 83%yield (29.77 g). ¹H NMR (CDCl₃) δ 7.4-7.2 (m, 12H), 6.9-6.8 (m, 2H),5.05 (AB quartet, 2H), 4.47 (d, J=8.5, 1H), 3.9-3.3 (m, 3H), 3.05 (d,J=8.5, 1H), 3.0-2.8 (m, 2H), 2.3-2.2 (m, 1H), 1.85-1.7 (m, 1H),1.45-1.15 (m, 4H) ppm.

Intermediate 11(1R,2R,1″S)-1-(4-Benzyloxy-phenyl)-2-(2″-hydroxy-1″-phenyl-ethylamino)-3-pyrrolidin-1-yl-propan-1-ol

In a 3-neck flask with dropping funnel and condenser under nitrogen wasadded LiAlH₄ (Aldrich, 6.3 g, 166 mmol, 2.57 equivalents) and anhydroustetrahydrofuran (75 mL). A solution of Intermediate 10 (29.7 g, 64.48mmol) in anhydrous tetrahydrofuran (300 mL) was added dropwise to thereaction over 15-30 minutes. The reaction was refluxed under nitrogenfor 9 hours. The reaction was cooled in an ice bath and water (7.0 mL)was very carefully added drop by drop (vigorous exothermic reaction withhydrogen being given off). A 15% aqueous NaOH solution (7.0 mL) wasadded dropwise followed by water (21 mL). Halfway through the finalwater addition a large amount of a white solid formed. It was broken upby the addition of methylene chloride (250 mL). After stirring at roomtemperature for 15 minutes, the mixture was filtered through a celiteplug (17 cm in diameter by 1 cm in height). The precipitate was washedwith methylene chloride (2×250 mL). The filtrate was rotoevaporated toan oil. The oil was dissolved in 1M aqueous HCl (300 mL). This aqueouslayer was washed with tert-butyl methyl ether (2×200 mL). After coolingin an ice bath, 10 M aqueous NaOH (35 mL) was carefully added to theaqueous layer (final pH=14). The product was extracted three times withmethylene chloride (300 mL, 200 mL and 100 mL). After drying with sodiumsulfate, the solution was filtered and rotoevaporated to yield a whitesolid. After drying, the Intermediate 11 was obtained in 94% yield (26.9g). ¹H NMR (CDCl₃) δ 7.46-7.115 (m, 12H), 6.98-6.96 (m, 2H), 5.08 (s,2H), 4.49 (d, J=4.7, 1H), 3.70-3.65 (m, 1H), 3.60-3.55 (m, 1H),3.54-3.45 (m, 1H), 3.00-2.90 (m, 1H), 2.7-2.6 (m, 1H), 2.36 (br s, 4H),2.15-2.05 (m, 1H), 1.70 (br s, 4H) ppm.

Intermediate 12(1R,2R)-2-Amino-1-(4-benzyloxy-phenyl)-3-pyrrolidi-1-yl-propan-1-olHydrogen Chloride Salt

Intermediate 11 (26.9 g, 60.24 mmol) was dissolved in methanol (400 mL)and 1M aqueous HCl (130 mL) was added. After being placed undernitrogen, 20% palladium hydroxide on carbon (Pearlman's catalysis,Aldrich, 10.8 g) was added. The reaction was placed under nitrogen andthen under hydrogen by evacuation and filling to a balloon. The mixturewas stirred for 48 hours at room temperature under a hydrogen balloon.The reaction was placed under nitrogen and filtered through a pad ofcelite. The celite pad was washed with 10% water in methanol (250 mL)and water (50 mL). The solvent was removed by rotoevaporation andcoevaporation with toluene (3×100 mL). The foamy solid was dissolved inisopropanol (300 mL) at reflux. The solution was cooled to roomtemperature and tert-butyl methyl ether (550 mL) was added. Afterstirring at room temperature for 2 hours, the white solid was filteredand washed with tert-butyl methyl ether. After drying, Intermediate 12was obtained in ca 99% yield (18 g). ¹H NMR (DMSO-d6) δ 9.68 (brs, 1H),8.53 (br s, 2H) 7.24 (d, J=8.55 Hz, 2H), 6.80 (d, J=8.55 Hz, 2H), 4.72(d, J=7.0 Hz, 1H), 3.8-3.6 (m, 2H), 3.4-3.6 (m, 3H), 3.0-3.2 (m, 2H),2.7-2.5 (br s, 1H), 2.0-1.7 (br s, 4H) ppm.

Compound 13 (1R,2R)-Hexadecanoic acid[2-(4-benzyloxy-phenyl)-2-hydroxy-1-pyrrolidin-1-ylmethyl-ethyl]-amide

To Intermediate 12 (16.17 g 49.36 mmol) suspended in tetrahydrofuran(500 mL) was added triethylamine (28 mL, 4 equivalents). A solution ofPalmitic acid N-hydroxysuccinimide ester (Sigma, 19.2 g, 54.29 mmol)dissolved in tetrahydrofuran (125 mL) was added over 30 minutes undernitrogen at room temperature. The solution was stirred at roomtemperature for 18-20 hours. The white precipitate was removed byfiltration and the filtrate was rotoevaporated to a foamy off-whitesolid (35.5 g). The crude material was dissolved in methylene chloride(500 mL) and washed with water (100 mL) and saturated aqueous sodiumcarbonate solution (100 mL). After drying with sodium sulfate, thesolution was filtered and rotoevaporated to yield a off-white foamysolid (24.75 g). This material was recrystallized from 40% ethyl acetatein heptane (500 mL, hot filtration). Compound 13 was obtained in 61%yield (14.45 g) Analytical chiral HPLC showed this material to be 99.7%the desired R,R isomer. Analytical HPLC showed this material to be 99.6%pure. mp 95-97° C. ¹H NMR (CDCl₃) δ 7.15 (d, J=8.5 Hz, 2H), 6.70 (d,J=8.5 Hz, 2H), 6.0 (d, J=7.3, 1H), 4.96 (d, J=3.8, 1H), 4.3-4.2 (m, 1H),2.9-2.7 (m, 2H), 2.65-2.55 (m, 4H), 2.10 (t, J=7.5, 2H), 1.75 (br s,4H), 1.58-1.46 (m, 2H), 1.32-1.16 (m, 24H), 0.9 (t, J=6.7, 3H) ppm.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1-23. (canceled) 24: A method of preparing a compound of formula (2)

said method comprising reacting a compound of formula (1)

with pyrrolidine, followed by hydrolysis. 25: The method of claim 24,wherein the hydrolysis is carried out using methanol as solvent. 26: Themethod of claim 24, wherein the compound of formula (1) is prepared byreacting (5S)-5-phenylmorpholin-2-one with1,4-benzodioxan-6-carboxaldehyde. 27: The method of claim 26, wherein(5S)-5-phenylmorpholin-2-one is prepared by reacting a compound offormula (i)

with a compound of formula (ii)

28: The method of claim 24, further comprising reducing the compound offormula (2) to form a compound of formula (3)

29: The method of claim 28, wherein the reduction is carried out usinglithium aluminum hydride. 30: A method of preparing a compound offormula (3)

said method comprising reducing a compound of formula (2)

31: The method of claim 30, wherein the reduction is carried out usinglithium aluminum hydride. 32: The method of claim 30, wherein thecompound of formula (2) is prepared by reacting a compound of formula(1)

with pyrrolidine, followed by hydrolysis. 33: The method of claim 32,wherein the hydrolysis is carried out using methanol as solvent. 34: Themethod of claim 32, wherein the compound of formula (1) is prepared byreacting (5S)-5-phenylmorpholin-2-one with1,4-benzodioxan-6-carboxaldehyde. 35: The method of claim 34, wherein(5S)-5-phenylmorpholin-2-one is prepared by reacting a compound offormula (i)

with a compound of formula (ii)